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Electromagnetic Fields Mediate Efficient Cell Reprogramming
Into a Pluripotent State
Soonbong Baek1, Xiaoyuan Quan1, Soo-chan Kim2, Christopher Lengner3, Jung-
keug Park1 and Jongpil Kim1*
1 Lab of Stem Cells and Cell Reprogramming, Department of Biomedical Engineering,
Dongguk University, Seoul, 100‐273, Korea
2Department of Electrical and Electronic Engineering, Hankyong National University,
Kyonggi-do, 456-749, Korea
3Department of Animal Biology, School of Veterinary Medicine, University of
Pennsylvania
* Correspondence should be addressed to
Jongpil Kim, Ph.D.
Assistant Professor
Dept. of Biomedical Engineering
Dongguk University
Seoul, Korea
Tel: 82-02-2260-3371
Fax: 82-02-2260-8726
Email: [email protected]
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ABSTRACT
Life on earth is constantly exposed to natural electromagnetic fields (EMF) and it is
generally accepted that EMF may exert a variety of effects on biological systems.
Particularly, extremely low frequency electromagnetic fields (EL-EMFs) affect
biological processes such as cell development and differentiation, however, the
fundamental mechanisms by which EMF influences these processes remain unclear.
Here we show that that EMF exposure induces epigenetic changes that promote
efficient somatic cell reprogramming to pluripotency. These epigenetic changes
resulted from EMF-induced activation of the histone lysine methyltransferase Mll2.
Remarkably, an EMF-free system that eliminates earth’s naturally occurring
magnetic field abrogates these epigenetic changes, resulting in a failure to undergo
reprogramming. Therefore, our results reveal that EMF directly regulates dynamic
epigenetic changes through Mll2, providing efficient tool for epigenetic
reprogramming including the acquisition of pluripotency.
Keywords: Electromagnetic Fields, Cell Reprogramming, Epigenetic Changes
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Earth has a magnetic field that extends from its inner core to where it meets
the solar wind, protecting us from a stream of cosmic rays emanating from space 1.
Its magnitude at the Earth's surface ranges from 25 to 65 µT and all life on earth has
evolved in the presence of this field. In addition to this naturally occurring magnetic
field, humans are now exposed to electromagnetic field (EMFs) more than ever as
electrical technology becomes increasingly ubiquitous.2 Thus, there are growing
concerns in public health regarding the potential consequences of increasing
exposure to man-made electromagnetic fields.3-7 Epidemiological studies show that
exposure to EMF is associated with a risk of cancer and other diseases.8, 9 However,
this myriad of studies suggests that there is a general agreement on the effects of
EMF on biological systems.
For example, it has been suggested that extremely low frequency
electromagnetic fields (EL-EMFs) influence numerous types of changes in cells
including migration, cell differentiation, apoptosis and stress response.10-15 EL-EMFs
also arise at various stages of embryonic development, affecting morphology and
migration of embryonic cells.15-17 Furthermore, it has been reported that a specific
frequency of EMF promote osteogenic and neurogenic differentiation that has been
clinically applied in the repair of bone fractures and to promote wound healing.18-21
Taken together, these collective studies indicate that EMFs may be involved in
governing cell fate conversion, although direct evidence of EMF effects on cell fate
plasticity remains lacking.
To examine whether EMFs play critical roles in controlling cell fate changes,
we ask whether EMF exposure can influence epigenetic reprogramming by
facilitating cell fate conversion. Somatic cells can be reprogrammed to induced
pluripotent stem cells (iPSC) by the overexpression of defined factors, most notably
the four “Yamanaka factors”, Oct4, Sox2, Klf4, and c-Myc.22 Epigenetic
reprogramming embodies the most dramatic change of cellular identity, so this
system enables us to study the role of EMFs in governing epigenetic plasticity.
Gaining specific insight into how EMFs influence epigenetic identity will provide a
foundation for harnessing this phenomenon in directed differentiation protocols and
for therapeutic purposes.
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Here, we report that EMF exposure results in enhanced reprogramming
efficiency in somatic cells. We find that a specific frequency of EMF enhances
epigenetic changes during the reprogramming process via the induction of Mll2, a
histone lysine N-methyltransferase, which is known to contribute to the methylation
of histone 3 lysine 4(H3K4me3). Furthermore, we demonstrate a requirement for
EMF in the generation of induced pluripotent stem cells (iPS cells) as EMF inhibition
is sufficient to completely abrogate epigenetic changes and cell fate conversion in
these systems. Finally, we demonstrate that these EMF-free phenotypes can be
rescued by the overexpression of Mll2. Thus, our results suggest that Mll2 mediates
EMF induced reprogramming at the level of the epigenome to drive cell fate change.
These results provide a foundation for harnessing this phenomenon in directed cell
fate plasticity and therapeutic application.
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RESULTS
A specific frequency of EMF exposure enhances epigenetic reprogramming of
mouse somatic cells.
In 2006, Takahashi and Yamanaka achieved nuclear reprogramming of
somatic cells to pluripotency through the direct expression of defined transcription
factors.22 The generation of iPS cells via direct epigenetic reprogramming provides
critical evidence for the plasticity of the somatic epigenome. In order to examine
whether EL-EMF can directly influence changes in epigenetic identity, we
reprogrammed mouse somatic cells into iPSCs under EMF exposure (Figure 1A).
Initially, we transduced mouse tail tip fibroblasts (TTFs) harboring an eGFP reporter
at the endogenous Oct4 locus 23 with a polycistronic doxycycline (dox)-inducible
lentiviral vector expressing the Oct4, Sox2, Klf4, and c-Myc (OSKM) transcription
factors 24 along with a lentiviral vector constitutively expressing a modified reverse
tetracycline transactivator (M2rtTA) (Figure 1A). After infection, dox was introduced
and the culture was exposed to varying EL-EMF frequencies (10Hz, 50Hz, 100Hz
frequencies at 1mT intensity) (Figure S1A). Remarkably, EL-EMF treated cultures
exhibited an increase in colonies undergoing reprogramming as assessed by
alkaline phosphatase (AP) staining, with a maximum increase in efficiency observed
15 days after infection and exposure 50Hz, 1mT EL-EMF (Figure S1A). We
quantified the expression of pluripotency-related genes in these assays. EL-EMF
exposure during the 2 weeks of dox treatment resulted in a significant increase in the
expression endogenous Oct4, Sox2 and Nanog relative to controls (Figure 1B). The
agonistic effects of EL-EMF on pluripotency-related gene expression were specific to
cells undergoing reprogramming, as EL-EMF exposed cells not expressing the four
factors did not exhibit any induction of these transcripts (Figure 1B). Specific
induction of additional embryonic stem cell specific genes such as EsrrB, Fbx15,
Rex1 and Zfp296 was observed in EL-EMF-treated cultures (Figure S1B). We
confirmed the observed increase in the efficiency of iPSC generation using Oct4-
eGFP fibroblasts, where EL-EMF resulted in approximately 30 fold increase in the
number of Oct4-eGFP+ cells (Figure 1C and D). Additionally, we examine
reprogramming efficiency using Nanog-eGFP knock-in fibroblasts 25 and observed a
similar increase in iPSC cell generation with exposure to EL-EMF (50Hz, 1mT)
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relative to controls (Figure S1C). These findings provide clear evidence that
exposure to EL-EMF promotes somatic cell reprogramming into pluripotency.
After withdrawal of Dox on day 15, Oct4-eGFP-positive colonies were stable
and expressed pluripotency genes in a manner identical to mouse embryonic stem
cells (mESCs) after 50 passages, demonstrating that these iPSCs had reactivated
their endogenous pluripotency regulatory network (Figure S1D and Figure S1E). At
the protein level, all dox-independent iPSC lines stably expressed the pluripotency
markers Oct4, Nanog, Sox2 and SSEA-1 (Figure 1E), consistent with bisulfite
sequence analysis demonstrating demethylation of the endogenous Oct4 and Nanog
promoters in these iPS cells (Figure S2A). Since small molecules such as Valproic
acid (VPA) and Vitamin C have been reported to increase efficiency of iPSC
generation,26, 27 we compared reprogramming efficiencies between VPA, Vitamin C
and EL-EMF treated cultures. Strikingly, EL-EMF exposure resulted in the most
efficient iPSC formation confirmed by AP staining and Oct4-eGFP activity 15 days
after 4 factor induction (Figure 1F and Figure S2B). Teratoma formation analysis
confirmed the presence of cell types derived from all three embryonic germ layers
validating the pluripotency of these EL-EMF induced iPSCs (Figure S2C). Moreover,
we tested whether EL-EMF-induced iPSCs are germline competent. High
contribution chimeras were generated from these iPSCs and were crossed to
C57BL/6J mice. Resulting litters included agouti pups that, together with PCR
analysis, demonstrate the germline potential EL-EMF induced iPSCs (Figure S2D).
Taken together, these results demonstrate EL-EMF exposure in mouse somatic cells
results in significantly increased reprogramming efficiency.
Generation of iPS cells by Oct4 and EL-EMF exposure
This robust increase in reprogramming efficiency led us to examine whether
EL-EMF exposure could also replace some of the canonical reprogramming factors
during iPSC generation. To test this, we prepared dox inducible one factor (Oct4),
two factor (Oct4+Sox2) and control mouse embryonic fibroblasts (MEFs), in either
the presence or the absence of EL-EMF exposure 30 days after infection, we
observed that Oct4 and Sox2 combined with EL-EMF significantly increased iPSC
like colony formation indicating that EL-EMF can replace c-Myc and Klf4 efficiently
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(Figure S3A). Additionally, Oct4 alone combined with EL-EMF exposure induced AP
positive iPSC colonies after 30 days after infection (Figure 2A and 2B), whereas no
colonies were observed in the one or two factor infected conditions without EL-EMF
exposure (Figure S3A and Figure 2B). Additionally, we observed that treatment with
inhibitors of glycogen synthase kinase 3β (GSK3β) and mitogen activated protein
kinase (ERK1/2), known as 2i, in the Oct4 and EL-EMF-induced colonies lead to
further upregulation of pluripotency markers and decreases in H3K27me3 level in the
cells (Figure 2C and Figure S3B). 45 days after infection, all iPS colonies were AP+
(Figure 2B) and expressed the pluripotency markers Nanog, Oct4, and Sox2 (Figure
2D). Bisulfite sequencing of the Oct4 and Nanog promoters showed nearly complete
demethylation in Oct4/EL-EMF-induced iPSCs (OE-iPSCs) (Figure 2E). The genomic
integration of the Oct4 transgene was confirmed by PCR analysis (Figure 2F). These
cells were also competent in chimera formation assays (Figure S2D), demonstrating
their pluripotency and indicating that use of the EL-EMF exposure during
reprogramming has no adverse effect on the resulting iPSCs. Taken together, these
results demonstrate fibroblasts can be reprogrammed to pluripotency by the forced
expression of only one factor, Oct4, with EL-EMF exposure.
Mechanisms of EL-EMF induced cell reprogramming
To gain the insight into the mechanism by which EMF enhances
reprogramming, we first assayed global gene expression in cells undergoing
reprogramming in the presence of EL-EMF. To ensure rapid and homogenous
induction of cell reprogramming, we prepared inducible secondary MEFs in which all
of the cells in the culture harbor the identical site of integration and copy number of
dox-inducible reprogramming factors.28 Reprogramming of secondary MEFs was
initiated with dox in the presence or absence of EL-EMF exposure. To assess
changes in gene expression induced by EL-EMF, we analyzed the transcriptome of
secondary MEFs after EL-EMF exposure and compared the transcriptional profiles to
control secondary MEFs. EL-EMF exposure resulted in some changes in global gene
expressions (Figure 3A, Table 1). We identified 24 genes whose expression changed
after EL-EMF exposure and gene ontology (GO) analysis across the two
independent experimental sets identified an up-regulation of genes signatures
associated with cell cycle (Figure 3B). Quantitative RT-PCR analysis show that cell
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cycle related genes, including Ccnd1, Cyr61, Dazac1, Id3 and Nat5 were markedly
elevated in EL-EMF treated cultures in comparison to untreated cultures (Figure 3C).
However, these genes are expressed at the same level in ESCs and fibroblasts in
the presence or absence of EMF (data not shown). These results suggested that EL-
EMF exposure does not significantly affect pluripotent or somatic conditions but may
promote cell fate conversion during the reprogramming process. Consistent with this
idea, we observed a significant increase in cell proliferation upon EMF exposure in
the secondary MEFs, but no induction of cell proliferation in fibroblasts and mESC
(Figure 3D, 3E and 3F). A previous study has demonstrated that increasing
proliferation during reprogramming via p53 or p21 inhibition results in accelerated
reprogramming.29 However, the increase in proliferation alone is insufficient to
account for the effects of EL-EMF observed in our study as we found EL-EMF
capable of replacing Sox2, Klf4, and c-Myc in the reprogramming cocktail, and a
much greater increase in reprogramming efficiency than can be accounted for by the
observed increase in cell proliferation (up to 30 fold efficiency with less than 2-fold
increase in proliferation). Thus, we reasoned that EL-EMF enhance reprogramming
process through additional mechanisms.
Direct reprogramming occurs with massive changes to the epigenome,
including changes in histone modification and DNA methylation.30-32 Prior studies
demonstrated that EL-EMF exposure can affect chromatin modification.33, 34 Thus, in
order to examine whether EL-EMF exposure affects histone modifications during
reprogramming, we measured the expression level of histone modifiers in the EL-
EMF exposed secondary fibroblasts. Most histone modifiers were grossly unaffected
in EL-EMF-exposed secondary fibroblasts 3 days after dox treatment, however, we
observed a dramatic upregulation of the KMT2D gene that encodes the lysine-
specific methyltransferase Myeloid/mixed-lineage leukemia 2 (Mll2) in EL-EMF
exposed cells (Figure 4A and 4B). Induction of Mll2 precedes upregulation of Oct4
expression in the reprogramming process (Figure S4A), and Mll2 expression is
significantly increased at day 6 after dox treatment in the EL-EMF condition (Figure
S4A), indicating that Mll2 may facilitate pluripotency gene activation. Interestingly,
the upregulation of Mll2 and H3K4me3 levels was not observed in fibroblasts treated
with EL-EMF alone (Figure 4E, S4B, S4C and S4D)
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Mll2 is a member of trithorax (trxG) group that is mainly responsible for
H3K4 trimethylation (H3K4me3) during development.35 Thus, we measured the
enrichment level of histone modifications that mark active chromatin (H3K4me3) in
the EL-EMF exposed secondary fibroblasts. Surprisingly, consistent with previous
results, H3K4me3 accumulation was specific to cultures treated with EL-EMF
exposure, while H3K9me3 levels were not changed by EMF exposure alone or in
combination with 4 factor expression (Figure 4E and S4D). The accumulation of
H3K4me3 by EL-EMF exposure was much higher than in the 4 factor induced
reprogramming at each time points in the absence of EL-EMF, suggesting that EL-
EMF increase H3K4me3 levels, which then increases reprogramming efficiency
(Figure 4C, 4D, and S4E). We further found an increased interaction between Mll2
and H3K4me3 upon EL-EMF induced reprogramming (Figure 4F), indicating that
increased H3K4me3 level is a specific effect of EL-EMF exposure. To further assess
chromatin state at pluripotency-associated loci, we performed chromatin precipitation
(ChIP)-qPCR for these histone modifications. We observed that transcription start
sites of Oct4, Nanog and Esrrb are enriched for H3K4me3 in EL-EMF exposed
reprogramming cultures (Figure 4G). Taken together, these results suggest EL-EMF
induced H3K4me3 accumulation increases accessibility of several pluripotency-
associated loci during reprogramming.
Next, we investigated the consequences of increased Mll2 expression
during cell reprogramming. We confirmed that Mll2 overexpression led to a
significant induction in H3K4me3 levels during reprogramming (Figure S5A).
Moreover, we detected increased Oct4 and Nanog binding at the Nanog locus in
cultures overexpressing Mll2 at day 6 after dox treatment, and this binding could be
attenuated by Mll2 depletion (Figure 4H). Consistent with this, the number of alkaline
phosphatase colonies was markedly increased by Mll2 overexpression (Figure S5B).
Conversely, suppression of Mll2 during reprogramming led to reduction in the
number of Nanog-positive colonies (Figure S5B), suggested that that Mll2 governs
the epigenetic reprogramming efficiency of somatic cells.
To better understand the link between EL-EMF exposure H3K4me3 levels
and reprogramming efficiency, we examined the effects of EL-EMF on
reprogramming under condition of H3K4me3 inhibition. We observed that Mll2
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knockdown in EL-EMF induced reprogramming led to significant suppression of
global H3K4me3 level (Figure 5A), and reprogramming efficiency (Figure 5B).
Consistent with this result, the expression of Nanog was significantly decreased by
H3K4me3 inhibition on EL-EMF induced reprogramming (Figure 5C). Furthermore,
we observed that Mll2 inhibition in EL-EMF mediated reprogramming significantly
attenuates Oct4 binding at endogenous Oct4 and Nanog loci (figure 5D).
Additionally, we asked whether Mll2 mediated EL-EMF effects on the cell
cycle. Lentiviruss encoding Mll2 shRNA and Cre were introduced into MEF derived
from p53flox/flox mice.36 We found that p53 knockout (KO) and EL-EMF resulted in
significant increase in cell proliferation, inhibition of Mll2 coupled with p53KO or EL-
EMF had no negative effect on cell proliferation (Figure 5E). Despite the absence of
an effect of Mll2 inhibition on cell proliferation during reprogramming in the presence
of EL-EMF, Mll2 inhibition did significantly abrogate enhanced reprogramming
efficiency resulting from EL-EMF exposure (Figure 5F, 5G and S5C). Interestingly,
Mll2 inhibition had little negative effect on the increase in reprogramming efficiency
resulting from p53 inhibition, which is known to be a consequence of increased cell
cycling (Figure 5F, 5G and S5C).29 Taken together, these results strongly suggest
Mll2 mediated EL-EMF effects on cell reprogramming are cell cycle independent.
Cell reprogramming in a magnetic field-free system
The earth exists in a geomagnetic field and as such life on earth has evolved
in the presence of this magnetic field. It is therefore very difficult to estimate the
effects of the environmental magnetic field on biological systems. In order to
understand the basic role of EMF in biological systems, we devised a magnetic field-
free system in the culture incubator that maintains a zero magnetic field using a 3
axis helmholtz coil (Figure S6A and S6B).37 We put a 3-axis sensor in the center of a
helmholtz coil and a 3-dimensional EMF generator generated reverse magnetic
fields to keep a magnetic field-free space by adjusting the voltage across the coil
with a power supply (Figure S6C).
In our initial experiments, we prepared TTFs transduced with dox inducible
lentiviral vectors expressing OSKM and cultured them in the center of the 3 axis
helmholtz coil (Figure 6A). Expression of pluripotency-related genes including Oct4
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and Nanog was attenuated, and fibroblast-specific genes such as Dcn and Fap were
not efficiently suppressed in the EMF-free system (Figure 6B). Surprisingly, we found
that EMF-free environmental system greatly delayed generation of Oct4 and Nanog-
positive iPS colonies (Figure 6C). Next, we investigated histone methylation levels at
pluripotency loci in the EMF-free system. Interestingly, reprogramming in the
absence of EMFs resulted in significantly diminished H3K4me3 levels and slightly
impeded the reduction in H3K9me3 that is known to be a barrier for reprogramming
38 at pluripotency loci including Oct4, Nanog and Esrrb (Figure 6D and Figure 6E).
These results suggest that chromatin accessibility may be a rate-limiting factor for
reprogramming in the EMF-free environment. By extension, this result further
suggests that the environmental electromagnetic field is major factor for the process
of epigenetic reprogramming.
In light of these findings, we cultured control ES cells (V6.5) in the EMF-free
system to examine whether the absence of an EMF can influence the maintenance
of pluripotency. ES cells that were exposed to EMF-free conditions for 7 days were
viable and displayed typical ES cell morphology indistinguishable from control cells
(Figure S6A). Furthermore, the expression level of pluripotency genes in ES cells
was unaffected in the earth’s magnetic field cancellation system (Figure S7A and
S7B), suggesting that the pluripotent state was not affected by the absence of an
EMF. Additionally, fibroblasts grown in the EMF-free conditions were similarly
unaffected (Figure S7C). Additionally, expressions of cell cycle related genes are not
altered by EMF cancelation (Figure S7D).
We also observed the downregulation of Mll2 expression during iPS cell
generation in the EMF-free system (Figure 6F). This observation, coupled with our
prior data, suggests that the failure to induce Mll2 in the absence of EMF may
prevent reprogramming through inhibiting chromatin reorganization and accessibility
of the reprogramming factors to critical loci. Thus, we asked whether Mll2
overexpression could rescue cell reprogramming in the absence of EMF.
Overexpression of Mll2 rescued most of the defects associated in the EMF-free
system during reprogramming, increasing the number of iPS colonies (Figure 6G).
These results further supports a model in which the environmental magnetic field
promotes chromatin reorganization through the activation of Mll2 specifically during
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the dynamic epigenetic changes initiated by expression of the 4 YAMANAKA
reprogramming factors.
Conclusion
As one of the fundamental forces of nature, EMFs is a physical energy
produced by electrically charged objects that can affect the movement of other
charged objects in the field. Here we show that this physical energy can affect cell
fate changes and is essential for reprogramming to pluripotency. Exposure of cell
cultures to EMFs significantly improves reprogramming efficiency in somatic cells.
Interestingly, EL-EMF exposure combined with only one Yamanaka factor, Oct4, can
generate iPSCs, demonstrating that EL-EMF exposure can replace Sox2, Klf4 and c-
Myc during reprogramming. These results open a new possibility for a novel method
for efficient generation of iPSCs. Although many chemical factors or additional genes
have been reported for the generation of iPSCs, limitations such as integration of
foreign genetic elements or efficiency remain a challenge.39 Thus, EMF induced cell
fate changes may eventually provide a solution for efficient, non-invasive cell
reprogramming strategies in regenerative medicine.
Interestingly, our result show that ES cells and fibroblasts themselves are
not significantly affected by EMF exposure; rather, cells undergoing dramatic
epigenetic changes such as reprogramming seem to be uniquely susceptible to the
effects of EMFs. Consist with this, it has been shown previously that EL-EMF is
involved in the efficient neuronal development or other dynamic biological
processes.18, 20, 40 Consistent with this idea, we demonstrated that EL-EMF exposure
increases enrichment of H3K4me3 histone modification via Mll2 during development
and reprogramming and that this is closely associated with activation of gene
expression. Importantly, the suppression of epigenetic reprogramming in EMF-free
conditions indicates that EL-EMF energy is essential for favorable epigenetic
remodeling during the acquisition of pluripotency.
Furthermore, we found a specific induction of Mll2 by EL-EMF exposure
during reprogramming, which led to accumulation of H3K4me3, subsequently
facilitating robust Oct4 and Nanog occupancy at pluripotency-regulating loci,
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presumably in conjunction with the trxG complex. Thus, these results describe a
unique role for Mll2 in facilitating binding of the Yamanaka factors to target loci to
facilitate reprogramming (Figure S8A). Additionally, downregulation of Mll2
expression in the EMF-free system during reprogramming is intriguing because
these EMF-free phenotypes could be rescued by overexpression of Mll2. Thus,
taken together, these data provide strong support that Mll2 is a key mediator of the
effects of EMF during reprogramming. Future studies on the detailed mechanisms
through which Mll2 senses EMF will provide more insight into the relationship
between EMF and epigenetic plasticity.
Surprisingly, we found that an EMF-free environment was detrimental to cell
fate change of epigenetic reprogramming. The earth has a substantial magnetic field
and life on earth has evolved in the field of naturally occurring earth’s magnetic field.
It is possible that life may be have evolved to utilize this magnetic field to catalyze
biological processes. During cell fate determination of reprogramming, environmental
EMFs may be critical for the epigenetic changes to rapidly and precisely respond to
signals from the environment. Thus, these studies define the fundamental role of
EMFs in establishing cellular identity of cell reprogramming, and it will be of great
interest to understand the molecular mechanisms underlying the effects of EMFs on
the reprogramming in the future.
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Table 1. List of differentially expressed genes of secondary fibroblasts in the
presence and absence of EL-EMF
TargetID GeneSymbol Panther_Function
ILMN_2612325 4931406C07Rik Molecular function unclassified
ILMN_2846865 Actb Cytoskeletal protein -> Actin family cytoskeletal protein -> Actin and actin related protein
ILMN_2588055 Actb Cytoskeletal protein -> Actin family cytoskeletal protein -> Actin and actin related protein
ILMN_2774049 Amotl2 Miscellaneous function -> Other miscellaneous function protein
ILMN_2461696 Arfgap2 Nucleic acid binding;Select regulatory molecule -> G-protein modulator -> Other G-protein modulator
ILMN_2773862 C730025P13Rik Molecular function unclassified
ILMN_2669793 Ccnd1 Select regulatory molecule -> Kinase modulator -> Kinase activator
ILMN_1255422 Ccrn4l Nucleic acid binding
ILMN_2646209 Cdk10 Kinase -> Protein kinase -> Non-receptor serine/threonine protein kinase
ILMN_2603647 Clec2d Receptor -> Other receptor;Signaling molecule -> Membrane-bound signaling molecule;Defense/immunity protein -> Other defense and immunity protein
ILMN_2794645 Cyr61 Signaling molecule -> Growth factor
ILMN_2860674 Dazap1 Nucleic acid binding -> Other RNA-binding protein
ILMN_2678714 Id4 Transcription factor -> Other transcription factor
ILMN_2782082 Itm2b Miscellaneous function -> Other miscellaneous function protein
ILMN_2982200 Mrpl13 Nucleic acid binding -> Ribosomal protein
ILMN_1223543 Nat5 Transferase -> Acetyltransferase
ILMN_2744380 Npc2 Molecular function unclassified
ILMN_3116570 Pdhb Oxidoreductase -> Dehydrogenase
ILMN_2635895 Plscr2 Transfer/carrier protein -> Other transfer/carrier protein
ILMN_2753108 Surf6 Nucleic acid binding
ILMN_2602257 Tmem68 Molecular function unclassified
ILMN_1225522 Trpc4ap Molecular function unclassified
ILMN_1225360 Ythdf2 Molecular function unclassified
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Materials and Methods
EL-EMF exposure
We employed EL-EMF exposure system which was previously described.42 Briefly,
cells were continuously exposed, for up to 30-45 days, to EL-EMF (F=50Hz,
Bm=1mT) produced by a solenoid placed inside the Co2 incubator. The device was
supplied by an AC power supply (PCR-100L, KIKUSUI, Japan ), and EF frequency
and amplitude were monitored by an EF sensor (TM-701, Kanetec, Japan)
connected simultaneously to a microcontroller with ADC (Atmega328p, Atmel, USA).
Simulated magnetic flux density distribution by COMSOL 3.4 (COMSOL, MA) with
parameters as follows: axial symmetry (2D), radius (R ¼ 7.5cm), current (I ¼ 200
mA), and number of loops (N ¼1000). Control cells were grown in a different Co2
incubator under the same conditions without exposure to EL-EMFs. The geometry of
the system assured field uniformity for the exposed cultures. The surfaces of the
culture well plates were parallel to the force lines of the alternating magnetic field in
the solenoid. To exclude uncontrolled thermal effects of the field during the culture,
the maintenance of 37±0.1C inside each exposed well was controlled by direct
temperature measurement with thermometric probes. 1 day after infection, the
fibroblasts were cultured in the EMF incubator and the cultures were moved into the
normal incubator after iPSC formations.
3D Electromagnetic field Cancellation System
The Earth’s magnetic field is a natural component of the environment for living
organisms. The intensity of geomagnetic field is about 300-400mG. In order to
generate zero field environment by canceling the earth's magnetic field in the cell
culture incubator, we used 3-axis Helmholtz coils and the 3-axis magnetic sensor
(DC Miligauss Meter 3 axis, AlphaLab Inc, USA) which measures the earth field
accurately and the system is capable of canceling the earth field inside the 3-axis
Helmholtz coil. Helmholtz coils are used to generate uniform magnetic field and each
set of Helmholtz coils consists of two coils in a special arrangement to maximize the
spatial volume of uniform magnetic field. The magnetic field generated is
proportional to a DC or AC current into the coil which provide a calibration curve
between the field and the strength of DC current, called field-current calibration. 3-
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axis coil offer standard Helmholtz coils with different dimensions for generating
single axis, two-axis, or three-axis magnetic field and a 3-dimensional EMF
generator generated reverse magnetic fields to keep a magnetic field-free space by
adjusting the voltage across the coil with a power supply (PS2520G, Tektronix, USA).
The cell cultures were located in the center of the 3-axis Helmholtz coil in the
incubator and control cells were grown in a different Co2 incubator under the same
conditions without Helmholtz coil.
Cell culture
HEK293 cells were used for packaging the virus. These cells were grown in
fibroblast media (High glucose DMEM[Invitrogen], 10%FBS[Hyclone] and
5%penicilin/streptomycin[Invitroge]). These cells were co-transfected with the
lentivirus construct, psPAX2, pMD2.G and tetO-OSKM/FUW-M2rtTA and OKSIM,
vectors using calcium phosphate co-precipitation. Cell culture medium was replaced
24 h after transfection and virus harvested 72 h later. Mouse fibroblasts and human
dermal fibroblasts were transduced (40,000 cells) at passage 2 or 3 in 6-well culture
dishes with lentivirus. Infected mouse fibroblasts were cultured in mESC media with
Dox(2ug/ml) and human fibroblasts were cultured in hESC media.43 The ‘2i’ including
ERK(PD0325901) and GSK3b inhibitor (CHIR99021) were purchased from LC biolab.
VPA was purchased from Sigma -Aldrich. Drugs working concentration:
bVPA(1mM),Vc(30ug/ml),PD0325901(1uM),CHIR99021(3uM).
Alkaline phosphatase staining
Alkaline phosphatase staining was performed using an Alkaline Phosphatase
substrate kit (Millipore) according to manufacturer’s recommendations. For the
number of AP+ colonies, equal numbers of cells were plated on 100-mm dishes
coated with gelatin. Experiments were repeated three times, and data represented
the mean of triplicate wells ± SEM.
Immunofluorescence analysis
iPS cells were cultured on pretreated coverslips, fixed with 4% PFA. The cells were
then stained with primary antibodies against human/mouse Oct4 (Santa Cruz) and
mouse Nanog (Bethyl Lab) and H3K4me3 (Abcam) and H3K9me3 (Abcam) and
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H3K27me3 (Millipore) and human TRA-1-60(Millipore) and human/mouse
Sox2(R&D)and human Nanog (R&D). Respective secondary antibodies were
conjugated to Alexa Fluor (Invitrogen). Nuclei were counterstained with 4,6-
diamidino-2-phenylindole(DAPI; Invitrogen). Cells were imaged with Nikon eclipse Ti.
The two color Images were saved as a tif file format and merged it with Adobe
Photoshop software.
Flow cytometry
All flow cytometry was performed on a C6 cytometry (Accuri). Data were analyzed
with FlowJo software (TreeStar). Briefly, cells were dissociated with trypsin for 5
minutes and single cells were then pelleted, resuspended in ice-cold 4%
paraformaldehyde, and incubated for 10 min at 4°C. The cells were washed twice,
resuspended in FACS buffer for analysis on a FACS analysis.
Western blot
Western blot was carried out as described previously.44
PCR analysis
For quantitative PCR (qPCR) analysis, RNA was isolated using purelink RNA mini kit
(Ambion). Complementary DNA was produced with the Super Script III kit
(Invitrogen). Real-time quantitative PCR reactions were set up in triplicate with the
SYBR FAST qPCR Kit(KAPA), and run on a step one plus realtime PCR system
(Applied biosystem). Gene expression data were presented as relative expression to
GAPDH. Primer sequence: previously described Mouse primer sequence 22was
employed. Human primers were purchased from Allele iPSC RT-PCR primer set
(CAT# ABP-SC-iPShRES)
Chromatin immunoprecipitation (ChIP)-qPCR
2’MEFs, reprogramming intermediates, iPS cells (7 × 106) were collected for ChIP
analysis performed using the Enzymatic chromatin IP kit (Cell signaling) per the
manufacturer’s instructions. Primers Sequence referred in previous report.45
Bisulphite sequencing.
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Bisulphite sequencing was performed according to previously described42, 46. Briefly,
Bisulfite reactions were carried out according to the manufacturer’s instructions
(Epitect Bisulfite Kit, Qiagen). 2–4 µl of bisulfite treated DNA was used in a standard
PCR protocol to amplify Oct4 and Nanog promoter regions in mouse V6.5 ES cells,
fibroblast and iPS cells. PCR products were cloned into pCR2.1 vector (Invitrogen)
and sequenced using the M13
Generation of teratomas and chimaeras.
iPS cells were collected and separated from feeders by sedimentation of iPS cell
aggregates. Cells were washed, resuspended in 500 µl mouse ES cell medium and
injected subcutaneously into SCID mice (Taconic). 4 weeks after injection tumors
were removed from euthanized mice and fixed in formalin. Samples were paraffin
imbedded, sectioned and analyzed on the basis of hematoxylin and eosin staining.
For blastocyst injections, iPSCs were injected into B6XDBA F2 host blastocysts as
described previously. 46
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Acknowledgments
This work was supported by the National Research Foundation of Korea funded by
the Ministry of Education, Science, and Technology (NRF-2009-0082941, NRF-
2013R1A1A1058835, NRF-2013M3A9B4076485, NRF-2013M3A9B4044387), Korea
Health Technology R&D Project, Ministry of Health & Welfare (HI13C0540) and the
Next-Generation BioGreen 21 Program, Rural Development Administration
(PJ009073).
Supporting Information Available: Supplementary Figure 1: EL-EMF exposure
enhances cell reprogramming. Supplementary Figure 2: Characterization of EL-EMF
induced iPSCs. Supplementary Figure 3: Characterization of Oct4 & EL-EMF
induced iPSCs. Supplementary Figure 4: Histone modifications by EL-EMF exposure
during reprogramming. Supplementary Figure 5: Mll2 facilitates efficient cell
reprogramming. Supplementary Figure 6: EMF cancellation system. Supplementary
Figure 7: Somatic cell reprogramming under EMF free system. Supplementary
Figure 8: Schematic representation of EL-EMF mediated reprogramming. Somatic
cell reprogramming under EMF free system. This material is available free of charge
via the Internet at http://pubs.acs.org.
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Figures
Figure 1. Efficient generation of iPS cells with EL-EMF exposure in mouse somatic
cells. (A) Schematic drawing of the cell reprogramming with EL-EMF exposure. (B)
RT-PCR analysis of pluripotency markers, Oct4, Sox2 and Nanog of OSKM infected
fibroblasts in the presence and absence of EL-EMF at 6 and 15 days after dox
induction. Three independent experiments of three sets each were performed. Data
represent mean ±SEM. Student t-test, **p<0.01. (C) GFP expression in iPSC
colonies generated from Oct4-eGFP Knock-in(KI) fibroblasts in the absence and
presence of EL-EMF exposure 15 days after dox treatment (upper panel: bright field,
lower panel: fluorescence photograph). Scale Bars: 100µm. (D) FACS analysis for
Oct4-GFP+ cells from Oct4-eGFP KI fibroblasts in the absence and presence of EL-
EMF exposure at 15 days after dox treatment. (E) Immunostaining of EL-EMF
induced iPSCs (E-iPSC) for the pluripotency markers Oct4, Sox2, Nanog and
SSEA1. Scale Bars: 100µm. (F) The representative image(Left) and number of GFP
positive colonies(Right) from 4 factor reprogramming combined with different
conditions: Valproic acid(VPA), Vitamin C(Vc) and EL-EMF exposure. Equal numbers
of 4 factor infected cells were plated but treated with the different conditions, EL-
EMF exposure result in the highest induction of AP+ cells at day 15. Scale Bars:
100µm.
Figure 2. Generation of iPS cells by Oct4 with EL-EMF exposure. (A) Schematic of
strategy of reprogramming by Oct4 alone with EL-EMF (B) Morphology and AP
staining of Oct4 and EL-EMF and Oct4 induced iPSCs, compared with Oct4 infected
fibroblasts, with AP staining 30 and 45 day after infection. Three independent
experiments of three sets each were performed. Scale Bars: 100µm. (C) Expression
levels of pluripotency markers (Oct4, Sox2, Nanog and Esrrb) in Oct4 & EL-EMF
induced iPS cells and Oct4 infected fibroblasts. Three independent experiments of
three sets each were performed. Data represent mean ±SEM. (D)
Immunofluorescence staining for pluripotency markers (Oct4, Sox2 and Nanog) of
Oct4 & EL-EMF induced iPSCs and Oct4 infected fibroblasts. Scale Bars: 100µm. (E)
Bisulfite sequencing of Oct4 and Nanog gene promoters showing the methylation
state of Oct4/EL-EMF induced iPSCs. Open circles indicate unmethylated, and filled
circles indicate methylated CpG dinucleotides. (F) Transgene integrations of Oct4 &
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EL-EMF induced iPSCs.
Figure 3. Gene expression analysis of EL-EMF exposure reprogramming (A)
Heatmap of differentially expressed genes in the presence and absence of EL-EMF
exposure during cell reprogramming. Each column represents experimental samples
and each row represents a gene. Control (+4F) indicates reprogramming in
secondary fibroblasts, and EL-EMF (+4F) indicates reprogramming in secondary
fibroblasts plus EL-EMF exposure. Expression differences are shown in different
colors. Red means 1.5 fold higher expression and green means 1.5 fold lower
expression. (B) GO analysis of differentially expressed genes affected by EL-EMF
exposure. (C) qRT-pcr analysis of cell cycle related genes, Ccnd1, Cybrb1, Dazac1,
Id4, and Nat5, Expression levels were normalized to GAPDH. Data are presented as
mean ± SEM; n = 3. (D) Growth curves for secondary fibroblasts in the presence or
absence of EL-EMF exposure. Three independent experiments of three sets each
were performed. Data represent mean±SEM. Student t-test, **p<0.01. (E) MTT
assay for cell number of control fibroblasts, and secondary fibroblasts during the
reprogramming process in the presence and absence of EL-EMF exposure. Three
independent experiments of three sets each were performed. Data represent
mean±SEM. (F) Growth curves for ESCs (V6.5) in the presence or absence of EL-
EMF exposure.
Figure 4. EL-EMF exposure during reprogramming promotes histone modifications
by Mll2. (A) qRT-PCR analysis of genes related to histone modification in the
presence and absence of EL-EMF exposure at 3days after dox treatment.
Expression levels were. Two independent experiments of three sets each were
performed. Data are presented as mean ± SEM; n = 5. (B) Western blot for Mll2 and
H3K4me3 in OSKM infected fibroblasts in the presence and absence of EL-EMF
exposure. (C) Western blot analysis for H3K4me3 and H3K9me3 in OSKM infected
fibroblasts at 5, 10 and 15days after infection. (D) Semiquantitative analysis of
H3K4me3 levels in OSKM infected fibroblasts at 5,10, and 15days after infection.
The western blot band intensities were measured with ImageJ software. H3K4me3
levels were normalized to β-actin levels. Three independent experiments of three
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sets each were performed. Data represent mean±SEM. (E) Immunofluorescence
images of histone modification markers (H3K4me3, and H3K9me3) in control
fibroblasts, and secondary fibroblasts in the presence and absence of EL-EMF
exposure. EL-EMF exposure in secondary fibroblasts leads to significant
accumulations of H3K4me3 level during the reprogramming. Scale Bars: 100µm. (F)
Coimmunoprecipitation of the Mll2 and H3K4me3 complex. Coimmunoprecipitation
studies carried out with lysates prepared from the secondary fibroblasts at 10days
after dox treatment. (G) H3K4me3 and H3K9me3 Chip-PCR at Oct4, Nanog and
EsrrB promoters in secondary fibroblasts at 7 days after dox in the absence and
presence of EL-EMF exposure. Three independent experiments of three sets each
were performed. Data represent mean±SEM. (H) ChIP-qPCR of Oct4 and Nanog at
Nanog locus in reprogrammed cells, reprogramming cells combined with Mll2
overexpression and Mll2 knockdown. Three independent experiments of three sets
each were performed. Data represent mean±SEM.
Figure 5. Mll2 mediated EL-EMF effects on reprogramming. (A) Western blot for
H3K4me3 in EL-EMF induced reprogramming in the absence and presence of Mll2
inhibition. H3K4me3 in EL-EMF mediated reprogramming was repressed by Mll2
knockdown. (B) Representative image (Top) and numbers (bottom) of Oct4-GFP
colonies in EL-EMF induced reprogramming in the absence and presence of Mll2
inhibition. EL-EMF mediated reprogramming efficiency is significantly decreased by
Mll2 knockdown. Three independent experiments of three sets each were performed.
Data represent mean±SEM. Scale Bars: 100µm. (C) mRNA expression of Nanog
in EL-EMF mediated reprogramming coupled with Mll2 knockdown. Three
independent experiments of three sets each were performed. Data represent
mean±SEM. (D) ChiP-qPCR of Oct4 mark at Oct4 and Nanog loci in EL-EMF
mediated reprogramming coupled with Mll2 Knockdown. Three independent
experiments of three sets each were performed. Data represent mean±SEM. (E)
Growth curves of OSKM infected fibroblasts in the absence and presence of EL-EMF
exposure coupled with p53 knockout and Mll2 knockdown. Three independent
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experiments of three sets each were performed. Data represent mean±SEM. (F) The
number of Nanog positive colonies from OSKM infected fibroblasts in the absence
and presence of EL-EMF exposure coupled with p53 knockout and Mll2 knockdown
at 20days after OSKM infection. Three independent experiments of three sets each
were performed. Data represent mean±SEM. (G) The number of Nanog positive
colonies derived from 105 fibroblasts in the absence and presence of EL-EMF
exposure coupled with p53 knockout and Mll2 knockdown at 20days after OSKM
infection. Three independent experiments of three sets each were performed. Data
represent mean±SEM.
Figure 6. The earth’s magnetic field free system inhibits epigenetic reprogramming.
(A) Schematic drawing of the cell reprogramming under EMF free system. (B)
Quantitative expression levels of pluripotency genes (Oct4, Nanog) and fibroblast
specific genes (Dcn, Fap) in cultures reprogrammed with 4-factors in the presence
and absence of EMF. Three independent experiments of three sets each were
performed. Data represent mean±SEM. (C) Immunofluorescence staining for
pluripotency markers, Oct4 and Nanog, reprogrammed with 4 factors in the presence
and absence of environmental EMF at Day15 after dox treatment. Scale Bars:
100µm. (D and E) H3K4me3(D) and H3K9me3 (E) Chip-qPCR at pluripotency loci,
Oct4, Nanog and Esrrb in control fibroblasts and 4factor induced fibroblasts at 7
days after dox induction in presence and absence of environmental EMF. Three
independent experiments of three sets each were performed. Data represent
mean±SEM. (F) mRNA expression of Mll2 under normal condition and EMF free
condition during reprogramming. Three independent experiments of three sets each
were performed. Data represent mean ±SEM. Student t-test, **p<0.01. (G) (Top)
Representative image of AP staining reprogrammed cells at 20 days after OSKM
infections with EMF free and EMF free/Mll2. (Bottom) Number of AP positive colonies
in the EMF free and EMF free+ Mll2 during reprogramming. Overexpression of Mll2
rescues EMF free phenotypes in the reprogramming. Three independent
experiments of three sets each were performed. Data represent mean ±SEM.
Student t-test, **p<0.01. Scale Bars: 100µm
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Oct4-eGFP KI
tail-tip fibroblasts iPSCs
TetO-OSKM
+M2rtTa
+Dox
- Dox
50Hz, 1mT
EL-EMF
Day15 Day1
GFP
Control 0%
4F 1.73%
4F+EL-EMF 60.9%
A
Day0 Day15 C D
E F
SSEA-1
Rela
tive e
xpre
ssio
n t
o G
APD
H
0
10
20
30
D0 D8 D15
Endo-Oct4
0
5
10
15
20
25
D0 D6 D15
Endo-Sox2
0
10
20
30
D0 D6 D15
Nanog
Days
**
** **
Nanog
SSEA-1
Nanog SSEA-1 Sox2
DAPI DAPI
DAPI DAPI
Oct4 Nanog
B
Figure 1.
Control VPA Vitamin C EL-EMF (4F) (4F) (4F) (4F)
0
500
1000
1500
2000
2500
AP+
colo
nie
s/pla
te
+4factor
Phase
G
FP
Ctrl 4F
Ctrl Ctrl EL-EMF 4F
Ctrl
4F +EL- EMF
4F +EL-EMF EL-EMF
EL-E
MF induce
d iPSCs
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Oct4
mE
SC
fi
bro
bla
sts
Nanog
OE
-iP
SC
Sox2 Oct4 DAPI Nanog
Nanog Sox2 Oct4 DAPI DAPI
DAPI
DAPI
Phase
Phase Contr
ol(+
Oct
4)
EL-E
MF (
+O
ct4)
DAPI
A B
C
D
Contr
ol
(+O
ct4)
D0
EL-E
MF
(+O
ct4)
D30
D45(+2i)
fibroblasts iPSCs
TetO-Oct4
+Dox
50Hz, 1mT
EL-EMF
Day30 Day1
E
Figure 2.
+Dox
GAPDH
4F- iPSCs Fibro.
Oct4- iPSCs
DW
Tg-Oct4
Tg-Sox2
Tg-Klf4
Tg-cMyc
F
0
50
100
150
200
D0 D30 D45
Fold
changes
Endo-Oct4
0
10
20
30
D0 D30 D45
Fold
changes
Sox2
0
10
20
30
D0 D30 D45
Fold
changes
Nanog
0
50
100
150
200
D0 D30 D45
Fold
changes
Esrrb
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0
20
40
60
80
D0 D2 D4 D6
Rela
tive A
sorb
ance
(570nm
)
Days
Control
EL-EMF
4F
4F+EL-EMF
Figure 3.
1
1
1
1
1
2
3
3
2
3
3
4
5
4
5
7
0 2 4 6 8
Carbohydrate metabolism
Cell adhesion
Oncogenesis
Other metabolism
Sensory perception
Nucleoside, nucleotide…
Signal transduction
Protein metabolism and…
Cell proliferation and…
Immunity and defense
Lipid, fatty acid and…
Intracellular protein traffic
Developmental processes
Transport
Cell structure and motility
Cell cycle
0
1
2
Rela
tive e
xpre
ssio
n
Ccnd1
0
1
2
3
Rela
tive e
xpre
ssio
n
Nat5
0
1
2
3
Rela
tive e
xpre
ssio
n
Cyr61
0
1
2
3
Rela
tive e
xpre
ssio
n
Dazap1
0
1
2
Rela
tive e
xpre
ssio
n Id4
A B
0
20
40
60
80
D0 D2 D4 D6
Cell n
um
bers
(10
5)
Days
Control(+4F)
EL-EMF(+4F) **
**
0
10
20
30
40
D0 D2 D4 D6
ES c
ell n
ubers
(10
5)
Days
Control
EL-EMF
C
D E F
-2 -1 0 1 2 3
4931406C07Rik
Actb
Actb
Amotl2
Arfgap2
C730025P13Rik
Ccnd1
Ccrn4l
Cdk10
Clec2d
Cyr61
Dazap1
Id4
Itm2b
Mrpl13
Nat5
Npc2
Pdhb
Plscr2
Surf6
Tmem68
Trpc4ap
Ythdf2
Fold change (4F+EL-EMF/4F)
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0
1
2
3
4
Mll1 Mll2 Set1a Set1b Prdm2 Wdr5 Kdm1a Kdm4b Dot1l
Rela
tive e
xpre
ssio
n t
o
GAPD
H
4F 4F+EL-EMF
Mll2
GAPDH
Normal
EL-EMF
6days after infection
Input IP:H3K4me3
IP:Mll2
H3K4me3
Mll2
4F 4F+EL-EMF 4F 4F+EL-EMF
IgG
4F+EL-EMF
0
2
4
6
Anti-Oct4 Anti-Nanog
Rela
tive e
nrich
ment to
GA
PD
H
Nanog locus fibroblast
4F
4F+Mll2 O/E
4F+Mll2 K/D
Figure 4. A
B
D
E
F
H
0
1
2
3
4
5
6
0 5 10 15
Rela
tive inte
nsi
ty
Days
H3K4me4
4F
4F+EL-EMF
G
0
1
2
3
4
5
Rela
tive e
nrich
ment
to G
APD
H
Oct4 Nanog Esrrb
■H3K4me3 □H3K9me3
Oct4-GFP
Oct4-GFP Oct4-GFP
C H3K4me3 H3K9me3 β-actin
0Days
5Days
10Days
15Days
4F 4F+EL-EMF
Oct4-GFP
H3K4me3 DAPI H3K9me3 DAPI
H3K4me3 DAPI H3K9me3 DAPI
H3K4me3 H3K9me3 DAPI DAPI
H3K4me3 H3K9me3 DAPI DAPI
Norm
al
4F+
EL-E
MF
EL-E
MF
4F
4F 4F+EL-EMF 4F 4F+EL-EMF
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Oct
4
GFP
Phase
4F 4F+shMll2 4F+EL-EMF 4F+EL-EMF +shMll2
H3K4me3
β-Action
4F 4F+shMll2 4F+ EL-EMF
4F+EL-EMF +shMll2
0
5
10
15
20
25
30
35
0 3 6 9 12
Rela
tive e
xpre
ssio
n
Days
Nanog
4F
4F+EL-EMF
4F+EL-EMF+shMll2
A
C
B
D
F G
Figure 5.
E
0
1000
2000
3000
4000
5000
GFP
+ c
olo
nie
s/pla
te
0
10
20
30
40
50
60
Oct4 Nanog
Rela
tive e
nrich
ment to
GA
PD
H
IP: Anti-Oct4 fibroblast
4F
4F+shMll2
4F+EMF
4F+EMF+shMll2
4F+EL-EMF
4F+EL-EMF+shMll2
0
100
200
300
400
0 2 4 6
Cell N
um
ber(
10
4Cells)
Days
4F
4F+shMll2
4F+p53 KO
4F+p53 KO+shMll2
4F+EMF
4F+EMF+shMll2
4F+EL-EMF
4F+EL-EMF+shMll2
0 500 1000 1500 2000 2500
4F
4F+shMll2
4F+p53 KO
4F+p53 KO+shMll2
4F+EMF
4F+EMF+shMll2
Nanog+ colonies/plate
4F+EL-EMF+shMll2
4F+EL-EMF
0 50 100 150 200 250 300
4F
4F+shMll2
4F+p53 KO
4F+p53 KO+shMll2
4F+EMF
4F+EMF+shMll2
Nanog+ colonies/105Cells
4F+EL-EMF+shMll2
4F+EL-EMF
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Figure 6.
A
0
1
2
3
4
5
Oct4 Nanog Esrrb
Rela
tive e
nrich
ment
to G
APD
H
H3K4me3 fibroblast
4F
4F+EMF free
-120
-90
-60
-30
0
30
60
90
120
Rela
tive
exp
ress
ion fibroblast
4F
4F+EMF free
B
0
1
2
3
4
5
6
Oct4 Nanog Esrrb
Rela
tive e
nrich
ment
to G
APD
H
H3K9me3 fibroblast
4F
4F+EMF free
iPSCs Mouse
fibroblasts
Dox OSKM
+M2rtta
EMF free
Day15 Day1
(3 axis helmholtz coil)
0
30
60
90
120
150
180
Control EMF free EMF
free/Mll2
AP+
colo
nie
s/pla
te
**
+4F
Control EMF free EMF free /Mll2
**
+4F
0
0.0005
0.001
0.0015
D0 D10+EMF
free
D10 D10+EMF
free
Rela
tive e
xpre
ssio
n
Mll2
Contr
ol
Nanog Oct4 DAPI DAPI
EM
F f
ree
Phase
Nanog Oct4 DAPI DAPI Phase
C
D
E
F
G
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Sox2 Nanog Nanog Sox2
Table of contents
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